The very strong interaction of biotin with the proteins avidin and streptavidin renders biotin-containing compounds useful for numerous applications. For example, many diagnostic tests use biotinylated derivatives. The widely used enzyme linked immunosorbent assay (ELISA), which was developed as an alternative to radioimmunoassays, employs biotinylated antibodies. Other biotinylated compounds have been used as probes and biotinylated nucleic acids have also been widely used. Purification techniques such as affinity chromatography frequently employ biotinylated materials.
More recently, biotin derivatives have been used in diagnosis and therapy of human disease. Notably, investigators have shown that use of a combination of monoclonal antibodies, streptavidin and/or avidin, and radiolabeled biotin derivatives, improves the diagnostic and therapeutic characteristics of the radiolabeled monoclonal antibody tumor targeting system. An example would be the “pre-targeting” of monoclonal antibody conjugates using the biotin/(strept)avidin ligand/anti-ligand pair for imaging and therapy of cancer.
One disadvantage of using biotin derivatives for many of these applications is their generally low solubility in aqueous media. In many examples, biotin derivatives and biotinylation reagents generally need to be solubilized in organic solvents or a medium comprising a substantial level of organic constituents to attain aqueous solubility. Insolubility of biotin derivatives and biotinylation reagents in aqueous solutions is particularly problematic for in vivo applications where organic solvents cannot be used.
It has been recognized that the availability of biotin derivatives for binding with avidin- and streptavidin-containing compounds may be greater when a spacer molecule is used between the biotin moiety and the other moieties to which the biotin is attached. The spacer molecules previously used have generally possessed low solubility in aqueous medium. Such spacer molecules reduce the aqueous solubility of the biotin-containing compound. In addition to causing solubility problems, the lipophilic nature of the biotin derivatives with spacer molecules of low water solubility may cause them to associate with blood components, rendering their biological half-life in in vivo applications longer than desired. Prior investigators have attached water solubilizing moieties to biotin and to compounds containing two biotin moieties, however, the prior art has failed to disclose or suggest biotin-containing compounds that comprise at least three (3) biotin moieties which are resistant to the enzyme biotinidase.
Heretofore, U.S. Pat. Nos. 5,541,287 and 5,578,287 disclose compositions for use in pre-targeted delivery of diagnostic and therapeutic agents, which compositions employ biotin/avidin as the ligand/anti-ligand binding pair. These patents disclose that the 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetra acetic acid (DOTA)-biotin adducts have desirable in vivo biodistribution and are cleared primarily by renal excretion. However, these patents only teach biotin-containing compounds of up to two (2) biotin moieties. These patents also report that the disclosed adducts are not stable in serum due to the presence of biotinidase. Poor in vivo stability therefore limits the use of such conjugates in therapeutic applications.
U.S. Pat. No. 5,326,778 discloses conjugates of biotin and deferoxamine for radioimmunoimaging and radioimmunotherapy, which conjugates are capable of binding metal ions to avidin or streptavidin. This patent fails to suggest a biotin-containing compound that contains at least three (3) biotin moieties and water-solubilizing moieties of 6 to 50 atoms in length.
U.S. Pat. No. 5,482,698 discloses a method for the detection and therapy of lesions with biotin/avidin polymer conjugates. The preferred polymers are the starburst dendrimers or dextrans. This patent, however, does not suggest water-soluble linker moieties of 6 to 50 atoms in length and biotinidase blocking groups in a discrete multi-biotin-containing compound. This reference also fails to suggest how dendrimers and dextrans can be used to produce discrete, water-soluble, multi-biotin-containing compounds.
U.S. Pat. No. 5,521,319 teaches that biotin compounds, which comprise water-soluble linkers, can be conjugated to various moieties; however, there is no suggestion that three (3) or more biotin moieties be present in the compound and that biotinidase blocking groups be incorporated to impart in vivo stability.
U.S. Pat. No. 5,750,357 describes a detectable synthetic copolymer wherein one monomer is a binding agent for microorganisms and the second monomer is a detectable label or a binding site for a detectable label, such as biotin. The polymerization of these two monomers results in a random co-polymer that may contain three (3) or more biotin moieties. However, there is no suggestion or disclosure that each biotin moiety be linked to a water-soluble moiety of 6 to 50 atoms in length, which in turn is linked to a cross-linker compound that has at least tri-functionality, such as benzene 1,3,5-tricarbonyl trichloride, starburst dendrimers, cascade dendrimers, polylysine, polyglutamic acid, and polyaspartic acid. The thermally initiated random polymerization disclosed produces a reaction mixture that consists of numerous polymeric species with varying molecular weights and number of biotin moieties. Such random biotin-containing compounds have a very limited utility in diagnostic and therapeutic applications. Further, this patent does not address the need for a biotinidase blocking moiety.
Hnatowich, et al., in “Investigations of Avidin and Biotin for Imaging Applications”, J. Nucl. Med., Vol. 28, pp. 1294–1302, No. 8 (1987), discloses a biocytin-DTPA-biotin dimer reaction product. This reference, however, does not suggest a compound comprising at least three (3) biotin moieties, water-soluble linker moieties of 6 to 50 atoms in length, and biotinidase protective moieties.
Biotinylated dextrans have been proposed as molecular probes by, for example, R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Edition, Chapter 15.5, Molecular Probes, Inc., Eugene, Oreg. These probes are not discrete molecular entities and do not address the need for resistance to biotinidase activity, nor do they suggest the use of water-soluble linker moieties of 6 to 50 atoms in length.
It is apparent, then, that the prior art has paid little attention to the water solubility of biotin-containing compounds, or the synthesis of biotin reagents having three (3) or more biotin moieties and resistance to biotinidase. Because biotin-containing compounds are of increasing interest for diagnostic and therapeutic applications, there is a need for enhanced biotin-containing compounds exhibiting excellent water solubility and improved resistance to cleavage by the serum enzyme biotinidase. The compounds according to the invention meet these needs, in part, through the selection of water-soluble linkers that allow for the optimum binding of the biotin moiety to avidin/streptavidin.